Calculation of calcium dynamics from single wavelength fura-2 fluorescence recordings

We describe techniques for measurements of cytosolic calcium dynamics in single current- or voltage-clamped nerve cells. The calculations of calcium dynamics are based on continuous recordings of fura-2 fluorescence intensity at one excitation wavelength after an initial reference measurement at two excitation wavelengths. We show that such single wavelength recordings are not only sufficient for the calculation of Ca2+ concentrations, but also lead to a superior signal-to-noise ratio at a high temporal resolution. Moreover, this strategy diminishes requirements for the experimental setup, such as a device necessary to switch quickly between excitation filters. We have applied this approach on measurements of cytosolic free Ca2+ in single-electrode voltage-clamped CA3 pyramidal cells in hippocampal slice cultures.